This is an Open Access article distributed under the terms of the Creative Commons Attribution License (
http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Cell therapy is a potential therapeutic approach for several neurodegenetative disease,
including Huntington Disease (HD). To evaluate the putative efficacy of cell therapy
in HD, most studies have used excitotoxic animal models with only a few studies having
been conducted in genetic animal models. Genetically modified animals should provide
a more accurate representation of human HD, as they emulate the genetic basis of its
etiology.

Results

In this study, we aimed to assess the therapeutic potential of a human striatal neural
stem cell line (STROC05) implanted in the R6/2 transgenic mouse model of HD. As DARPP-32
GABAergic output neurons are predominately lost in HD, STROC05 cells were also pre-differentiated
using purmorphamine, a hedgehog agonist, to yield a greater number of DARPP-32 cells.
A bilateral injection of 4.5x105 cells of either undifferentiated or pre-differentiated DARPP-32 cells, however, did
not affect outcome compared to a vehicle control injection. Both survival and neuronal
differentiation remained poor with a mean of only 161 and 81 cells surviving in the
undifferentiated and differentiated conditions respectively. Only a few cells expressed
the neuronal marker Fox3.

Conclusions

Although the rapid brain atrophy and short life-span of the R6/2 model constitute
adverse conditions to detect potentially delayed treatment effects, significant technical
hurdles, such as poor cell survival and differentiation, were also sub-optimal. Further
consideration of these aspects is therefore needed in more enduring transgenic HD
models to provide a definite assessment of this cell line’s therapeutic relevance.
However, a combination of treatments is likely needed to affect outcome in transgenic
models of HD.

Keywords:

Background

Despite very significant advances in understanding the causes of Huntington’s disease
(HD), an efficacious therapy remains elusive
[1]. Cell therapy is a putative treatment for HD that could slow down neurodegeneration,
replace lost cells and potentially provide a long-term benefit. Preclinical and proof-of-principle
clinical trials using fetal tissue grafts suggest that therapeutic benefits are possible
[2-4]. However, the usage of human fetal tissue grafts raises several ethical, logistical,
and safety concerns. Notably, the procurement of large quantities of human fetal tissue
at an appropriate developmental stage from elective abortions, establishing the absence
of genetic disease or any other potentially harmful contaminations, as well as the
heterogeneous (multiple donors) nature of the grafts, limit their potential usage
in a routine clinical setting
[5].

During the last decade, neural stem cell lines emerged as a potential alternative
to fetal tissue grafts, as they can be maintained and expanded in vitro. Human neural
stem cells (hNSCs) afford a sustainable and scalable homogenous cell source to treat
large cohorts of patients
[6]. There is evidence that cell therapy can slow down neurodegeneration and ameliorate
behaviour in rat models of Huntington’s disease
[2,7-9]. Replacement of lost cells is, however, a greater challenge. Although human neural
stem cells can differentiate into neurons after implantation
[10,11], improvements of functional deficits by fetal striatal transplants into a lesioned
rat striatum is associated with DARPP-32 neurons within the transplants
[12-14]. Despite good survival and differentiation of human neurons in the rats, differentiation
of cells into DARPP-32 neurons remains a challenge
[10,15]. Pre-differentiation of cells prior to implantation into a DARPP-32 phenotype therefore
could potentially result in an improved outcome
[16]. Proof-of-principle of this strategy for mouse embryonic and neural stem cells have
previously been demonstrated in rat or mouse excitotoxic models of HD
[9,17].

However, to successfully progress this approach to a routine clinical application,
it is essential to develop this approach for human stem cells
[18]. hNSC lines, such as the STROC05 cell line (derived from the ganglionic eminence
of a 12 week-old fetus) have the potential to differentiate in vitro into DARPP-32
cells
[19,20] and potentially could provide a source of pre-differentiated DARPP-32 neurons for
implantation. Ideally the potential efficacy of either undifferentiated or pre-differentiated
cells is evaluated in a genetic model that exhibits a progressive phenotype resembling
that of human HD. One of these models, the R6/2 transgenic mouse model (expresses
the exon1 of the human HD gene), is most commonly used to screen new therapies for
Huntington’s disease
[21]. The impact of undifferentiated and pre-differentiated STROC05 cells on behavioural
impairments and brain atrophy was therefore evaluated in the R6/2 mouse model of Huntington’s
disease.

Methods

Human neural stem cell line (STROC05)

The cmyc-ERTAM conditionally immortalized human striatal neural stem cell line (STROC05, kindly
provided by ReNeuron Ltd., Surrey, UK) was previously described
[19]. In brief, STROC05 cells were isolated from the whole ganglionic eminence of 12-weeks-old
human fetal brain. The cmyc-ERTAM gene was transfected into cells with the retroviral vector pLNCX-2 (Clontech). Transfected
cell colonies were isolated following neomycin selection before being expanded into
a clonal cell line
[22]. To maintain proliferation through the conditional immortalization gene, 4-hydroxy-tamoxifen
(4-OHT, 100 nM/ml; Sigma-Aldrich, UK) was added to proliferation media. The STROC05
cell line was expanded in T75 tissue culture flasks (Falcon, UK). Flasks were coated
with mouse laminin at a concentration of 1:100 (mouse, 10 μg/ml; Trevigen, USA) for
at least 2 hours at 37°C. Medium was changed every 2 days and cells were passaged
at 90% confluence. The expansion media consisted of Dulbecco’s Modified Eagle’s Medium/Ham’s
F12 (DMEM:F12; Gibco, UK) which was supplemented with additional components (Table
1). To stimulate proliferation, growth factors, such as basic fibroblast growth factor-2
(bFGF-2, 10 ng/ml; Peprotech, UK) and epidermal growth factor (EGF, 20 ng/ml; Peprotech,
UK), were added to the media.

Table 1.Composition of cell culture media to expand the STROC05 cell line

In vitro differentiation of STROC05 cells

To induce neuronal differentiation and increase the proportion of DARPP-32 cells,
STROC05 cells were grown in vitro for 21 days on laminin (mouse, 10 μg/ml, Trevigen) and poly-l-lysine (PLL, 100 μg/ml,
Sigma) coated T175 flasks with 90% confluence, as previously described
[20]. For the first week, differentiation was induced using media that contained all components
from the proliferation media, with the exception of bFGF-2, EGF and 6-OHT. For the
2nd and 3rd week of differentiation, media consisted of neurobasal media (Gibco) supplemented
with B-27 (Gibco), L-Glutamate (Sigma) and Purmorphamine. For the 2nd week of differentiation, bFGF was added again to the media as a survival factor
[23] and to promote a rostral positional specification of neurons
[24,25], but was omitted again for the 3rd week of differentiation as positional specification in most cells is completed. Purmorphamine
(1 μM, Calbiochem) was added to the culture media throughout the 3 weeks of differentiation.

Effect of harvesting on cell viability and differentiation

As differentiated cells are very vulnerable when removed from tissue culture flasks,
it is essential to establish whether harvesting these cells after long-term differentiation
affects their viability and differentiation status. For this, cells were harvested
with Trypzean EDTA for less than five minutes at 37°C, followed by adding a soybean
trypsin inhibitor to inactivate the enzymatic activity. After harvesting, cells were
centrifuged for 5 minutes at 1500 rpm and the cell pellet was re-suspended in 1 ml
of DMEM. Using the trypan blue exclusion test, cells were counted and viability was
established to be 89.5%. Cells were re-seeded on laminin-coated cover slips in 24
well plates at 100,000 cells per well. After 24 h, viability of these re-seeded conditions
was evaluated again using the live/dead stain (viability/cytotoxicity kit for mammalian
cells, Gibco) and compared to cells that were not harvested. For the live/dead stain,
media was aspirated and cells were washed once with PBS prior to incubation with 2
μM calceinAM (to detect live cells) and 4 μM ethidium homodimer-1 (EthD-1) (to detect
dead cells) in PBS (500 μL per well) for 45 minutes at 37°C. Photos were taken immediately
using a fluorescent microscope (Zeiss). A separate set of coverslips were fixed with
4% Parafix (Pioneer) for 5 min. Immunohistochemistry was used to establish if harvesting
of cells would affect the proportion of neurons (1:500, mouse anti-β-III-tubulin,
Tuj, AB7751, Abcam) and specifically DARPP-32 neurons (1:500, rabbit anti-DARPP-32,
AB1656, Chemicon) within the cell suspension. After overnight incubation (at room
temperature) with the primary antibody, an appropriate secondary ALEXA594 (1:1000,
Molecular Probes) was applied for 60 min prior to attaching the coverslips to microscopic
slides with Vectashield for fluorescence containing DAPI (Vector Laboratories). Total
DAPI, as well as Tuj and DARPP-32 cells, were counted under a Zeiss Axioscope.

R6/2 mice

All procedures of this study were carried out according to the UK Animals (Scientific
procedures) Act 1986 (PPL70/6445), as well as the ethical review process of King’s
College London. A widely used and well characterized mouse transgenic model of Huntington’s
disease, R6/2 mice present with a rapid disease onset that is evident as early as
6 weeks of age. Especially the development of a clear behavioural phenotype in the
R6/2 compared to the N171-82Q or HDH111 is important to establish a potential therapeutic efficacy.

The average life span of R6/2 mice with 210 CAG repeats is approximately 16 weeks
of age
[26]. Here, R6/2 mice were generated from a colony that was maintained by backcrossing
R6/2 males to (CBA × C57BL/6) F1 females (B6CBAF1/OlaHsd, Harlan, UK). Mice were kept
in standard housing conditions, on a standard chow diet with water available ad libitum. During the last 2 weeks of the study (12 and 13 weeks of age), a mash diet was prepared
by soaking chow pellets in water. These were placed in the floor of the cages within
easy reach of the motor impaired R6/2 mice. Transgenic mice were identified by Polymerase
Chain Reaction (PCR) on an ear tissue sample at 4 weeks of age, as previously described
[27].

Cell implantation

On the day of transplantation, the cells were harvested by incubation with Trypzean
EDTA for less than five minutes at 37°C, followed by adding soybean trypsin inhibitor
to inactivate the enzymatic activity. After harvesting, cells were centrifuged for
5 minutes at 1500 rpm and the cell pellet was re-suspended in 2 ml of DMEM for cell
counting. Cells were suspended in vehicle consisting of 2.5 ml of DMEM and 3.75 ml
of Hypothermosol (BioLife Solutions) at a concentration of 7.5 × 104 cells/μl. Using the trypan blue exclusion test, viability was determined to be 89%.

At 7 weeks of age, mice underwent stereotactic surgery for the injection of NSCs.
This allowed sufficient time to conduct pre-implantation MRI scans, as well as behavioural
test, after animals were weaned at 4 weeks of age from their mothers. Additionally,
animals’ genotype was determined and animals were randomly allocated to their experimental
groups based on a sequence of random numbers. Although at 7 weeks of age, R6/2 mice
do not exhibit a motor deficit
[28], they do nevertheless already show signs of brain atrophy
[29]. Impor-tantly, R6/2 mice do not display any neuronal loss
[28]. At this age, there is also a decrease, as well as morphological abnormalities, in
microglia
[30].

For cell implantation, anaesthesia was induced through isoflurane inhalation (Abbott)
at 4-5%, then maintained at 1.5-2%. Animals were mounted in a stereotaxic frame and
a sagittal incision was carefully made followed by the drilling of two burr holes.
Either 6 μl (3 μl per side, 0.5 μl/min) of cells or vehicle were injected with a 22
Gauge needle attached to 10 μl Hamilton syringe using a convection-enhanced delivery
[31] at Anterior-Posterior +0.5 mm (in relation to Bregma), Lateral ±2 mm and −3.5 mm
below the surface of the dura. The deposit was divided into two equal amounts; one
was injected at −3 mm (after retraction of the needle by 0.5 mm) and the other at
−2.5 mm. After injection, the syringe was left in place for 5 minutes and slowly withdrawn
over 3 minutes, followed by suturing of the incision. During the surgery, body temperature
was controlled using a homeostatic heating pad set at 37°C. No immunosuppression was
given as STROC05 cells exhibit a robust survival in the 3-nitropropionic acid rat
model of Huntington’s disease over 90 days (Additional file
1: Figure S1), as well as wild-type mice (Additional file
2: Figure S2).

Additional file 1.Figure S1. STROC05 survival in the 3-nitroproprionic acid (3-NPA) rat model of Huntington’s
disease. Male Lewis rats (220-250 g) received i.p. injections of 42 mg/kg 3-NPA. (Sigma-Aldrich)
for five consecutive days to induce a bilateral degeneration of striatal cells, as
previously described
[8]. Animals gradually develop a behavioural phenotype and show a progressive striatal
tissue loss that coincides with neuronal loss, as well as an increase in glial scarring
and microglia activity
[8]. Additionally, these animals show a clear deficit in brain activity
[7,60]. Two weeks after lesion induction, animals received unilateral injections of 400,000
STROC05 human neural stem cells (hNSCs). hNSCs can be detected in the injection tract
using human nuclear antigen (A). The presence of CD11b + microglia reveals the inflammatory
response to the ongoing neurodegeneration in the lateral striatum and indicates a
placement of cells just peripheral to the damage. Higher magnification images reveal
a limited migration from the injection tract to the area of damage (B&C). STROC05
cells retained some expression of nestin (D&E), but also partially differentiated
into GFAP + astrocytes. Using brightfield microscopy of cell survival (G) in animals
that were either immunocompetent or immunosuppressed using Cyclosporine A (CsA, Sandimmun,
Novartis, 10 mg/kg, diluted in Ringer’s solution) and methylpredinolone (20 mg/kg
day 1–7; 10 mg/kg day 8–12; 5 mg/kg day 13–14 i.p., Pharmacia Upjohn), a sterelogical
analysis indicated a robust cell survival under both conditions over 90 days. Over
10,000 cells survived in the immunocompetent group and 25,000 cells were present in
the immunosuppressed rats. It was only at 90 days survival that there was a significant
difference between immunosuppression and immunocompetent animals (* P < .05), but
there was no significant decrease in cell number between 30 and 90 days. Discontinuation
of immunosuppression also did not lead to a graft rejection.

Additional file 2.Figure S2. Acute survival of STROC05 in WT mice. An injection of 225,000 STROC05 cells in 3
μl (75,000 cells/μl) at 7 weeks of age into wild-type mice resulted in a good graft
survival (Human nuclei antigen, HNA, in red, DAPI in blue), even in the absence of
immunosuppression. Cells remained within the injection tract and did not exhibit any
migration out of their site of injection. To ensure a better distribution of cells
within the striatum, two deposits were placed within the same injection tract. A glial
reaction (GFAP + cells in green) was evident along the injection tract. These results
indicate that STROC05 cells can survive in WT animals and that using this protocol
there is a robust engraftment.

After surgery, post-operative care included fluid-replacement (0.1 ml saline/animal)
and a local analgesic (EMLA cream 5%; AstraZeneca, UK). The animals were singly caged
with softened food pellets and water available ad libitum for 24 h before being returned to their home cages.

Body weight

Weight loss is a prominent symptom in R6/2 mice
[28,32]. Body weight has often been used as a reliable outcome measure to assess the beneficial
effect of different therapeutic approaches in R6/2 mice
[26,33-37]. Mice were weighted weekly from the time of weaning (4 weeks) until the end of the
study. To avoid the impact of diurnal feeding habits, body weight was obtained weekly
on the same day and time.

Behavioural battery

For each behavioural test, the running order of animals was based on a randomization
of the cages, but within each cage (containing WT and R6/2), mice were run sequentially.
Animals within each cage were randomly chosen for each trial. If more than one trial
was conducted, this was run in the same sequence.

Rotarod

The rotarod is considered a very sensitive and reliable motor task to assess motor
coordination in HD transgenic mice
[26,38]. R6/2 mice are known to have impaired rotarod performance
[39,40]. According to a standard protocol
[26], mice were placed on a rotarod (Ugo Basile) with a 3 cm diameter rod at a constant
speed of 4 rpm for 20 sec. After this acclimatisation period, the rod speed accelerated
from 4 to 40 rpm over 300 sec. Latency for mice to fall from the rod was recorded.
Rotarod performance was assessed over three successive days with 3 trials per day.
The first assessment day was always excluded from analysis. Mice were tested one week
pre-transplantation, as well as at 1, 3, and 5 weeks post-transplantation.

Open field

The open field test has been used extensively as a reliable measure to evaluate locomotor
activity and anxiety-like behaviour in R6/2 mice
[41,42]. A custom-built 100 cm diameter and 35 cm deep circular open field arena (Engineering
& Design Plastics) was divided into outer and inner zones by a circle drawn 4 cm from
the outer walls. Mice were placed individually in the outer zone facing the centre
of the maze with their behaviour being automatically recorded by a camera for a period
of 5 min. Data was subsequently analysed using Ethovision XT7.0 software (Noldus).
The arena was cleaned between mice to prevent behavioural influences from the odours
of previous trials. Total distance travelled (locomotion) and time spent in the outer
zone (thigmotaxis, indicative of anxiety-like behaviour) were measured one week pre-transplantation,
as well as at 1, 3, and 5 weeks post-transplantation.

Grip strength

Grip strength analysis is a reliable and sensitive test to evaluate muscular strength
in R6/2 mice
[26,39,42]. To measure forelimb grip strength, mice were lowered towards the grid to grab it
with both front paws. Mice were gently pulled back until they released their grip
and the equipment automatically measured the force required to pry the mouse from
the grid. A single session consisting of 5 consecutive trials was recorded once a
week at 4, 5, and 6 weeks post-grafting. As low scores may be due to the mouse failing
to grip the grid effectively, the best three scores of the five trials were averaged.

Magnetic Resonance Imaging (MRI)

Six weeks following cell implantation, mice were anesthetised using isoflurane (4-5%
induction, 1.5-2% maintenance in 0.7 l/min medical air and 0.3 l/min oxygen) and fixed
within a head holder/respiration mask to reduce head movement. MR images were acquired
using a 7 Tesla magnet (Varian), equipped with a 100 Gauss gradient set and a 39 mm
transmission/receive coil (Rapid). A T2-weighted multi-echo multi-slice (MEMS) sequence was used (TR = 2500 ms, minimum TE = 10
ms, number of echo = 8, echo spacing = 10 ms, averages = 4, matrix = 128x128, and
FOV = 20 × 20 mm). Thirty coronal slices with 0.5 mm thickness were acquired across
the mouse brain. Manual segmentation of anatomical regions of interest (ROIs, Additional
file
3: Figure S3), including whole brain, striatum, cortex, hippocampus, and lateral ventricle,
was performed using JIM 5.0 (Xinapse). Criteria used to define ROIs are summarized
in Table
2. Manual segmentation of the same structure at two separate occasions yielded an intra-rater
discrepancy of less than 2% error.

Immunohistochemistry

After MRI scanning, anesthetized animals received an intracardial perfusion of saline
followed by 4% Parafix (Pioneer). Brains were excised and post-fixed for 24 h at 6°C
before being cryoprotected in 30% sucrose at 6°C. Sections (40 μm) were cut on a freezing
sliding microtome in the coronal plane and stored at −20°C in tissue cryoprotective
solution (25% glycerine, 30% ethylene glycol, and 50% PBS).

To identify transplanted cells, sections were stained with a mouse anti-human nuclear
protein (HNA) antibody (1:400, MAB1218, Millipore). For this, sections were rinsed
with PBS, blocked for 30 minutes in 0.1% H202 as inhibitor for endogenous peroxidase activity (Sigma), followed by 60 min incubation
in 10% blocking solution (10% normal goat serum in 0.3% Triton X-100 PBS) at room
temperature (RT, 21°C). To block the non-specific binding of endogenous biotin, the
sections were incubated with avidin-biotin blocking solutions (Vector) for 30 min.
The sections were incubated with the HNA antibody at RT for an hour, followed by 10
min of incubation at RT with secondary biotinylated anti-mouse antibody (1:200, Vector),
and 5 min at RT with an avidin-biotinylated-peroxidase complex (1:100 in PBS, Vector).
Secondary antibody binding was visualized using 3,3’-diaminobenzoic acid (DAB, Sigma)
dissolved in PBS with the addition of H202 to a concentration of 0.03% immediately before use. Finally, the sections were washed
in PBS, mounted onto glass slides, dehydrated for 5 min in each of 70, 85, 90, and
100% alcohol, cleared by xylene, and coverslipped with Entellen (Merck, UK).

Results

To assess whether transplantation of long-term differentiated cells is possible, long-term
differentiated cultures were harvested and reseeded to measure potential effects on
viability and neuronal differentiation. Viability straight after harvesting of differentiated
cells was above 90% as indicated by the trypan blue exclusion test. This good viability
was maintained after re-seeding these cells for 24 h (Figure
1A). The harvesting re-seeding procedure also did not reduce the neuronal population
(Figure
1B). The number of β–III-tubulin- and DARPP-32-positive cells remained fairly consistent
(Figure
1C). The number and percentage of astrocytes also was consistent between pre-harvesting
conditions and re-seeding (Figure
1D). These results suggest that the harvesting re-seeding process did not significantly
affect the viability of differentiated cells and the neuronal population is very similar
to the pre-harvest condition.

Cell implants do not impact on weight loss

Body weight is a reliable indicator of the overall health of R6/2 mice. The body weight
of wild type (WT) and R6/2 mice (n = 10/genotype) steadily increased until 8 weeks
of age (1 week post-implantation, Figure
2), after which they cease to gain weight. By 3 weeks post-implantation, R6/2 mice
had significantly lower body weight compared to WT mice. Animals that received undifferentiated
or differentiated cells followed the same weight pattern than those R6/2 mice that
received a vehicle injection. These results suggest that cell implantation did not
impact on weight loss in R6/2 mice.

Figure 2.Body weight. Weight gain between groups was equivalent up to 7 weeks of age when animals were
grafted. Post-implantation WT mice with vehicle injection continued to gain weight.
All R6/2 mice started to lose weight 3 weeks post-grafting (11 weeks of age). There
was no effect of the implantation of undifferentiated or differentiated cells on body
weight.

The development of a progressive behavioural phenotype is a key characteristic of
R6/2 mice. Up to 8 weeks of age (1 week post-implantation), the R6/2 animals performed
as well as WT controls on the rotarod (Figure
3A), but gradually thereafter their rotarod performance deteriorated as compared to
WT controls. Implantation of cells (undifferentiated or differentiated) did not prevent
this deterioration. A significant locomotor deficit was already evident in R6/2 animals
pre-implanted at 7 weeks of age (Figure
3B). This deficit gradually worsened and the cell therapy had no significant impact.
There was also no significant alteration in anxiety-like thigmotaxis behaviour in
the R6/2 mice (data not shown). Grip strength was consistently impaired in the animals
between 4 and 6 weeks post-grafting, and no improvement due to cell implantation was
evident (Figure
3C). Therefore, neither the bilateral implantation of undifferentiated, nor differentiated
cells significantly impacted on the emergence or the progression of clear behavioural
deficits in the R6/2 mouse model of Huntington’s disease.

Figure 3.Behaviour.A. Rotarod: no effect of genotype on rotarod performance was detected one week pre-
or post-grafting. However, a significant impairment was evident in R6/2 mice at 3
and 5 weeks post-grafting which was not improved through the implantation of undifferentiated
or differentiated cells. B. Open field: the total exploratory activity of R6/2 was reduced compared to WT controls
at all time points tested. Neither undifferentiated, nor differentiated, cells attenuated
the deterioration of R6/2 exploratory behaviour. C. Grip strength: all R6/2 mice showed significantly impaired performance compared to
WT mice at all time points. No beneficial effect of treatment was evident. (* p < 0.05,
**p < 0.01, and ***p < 0.001).

Survival and differentiation of cell implants

The survival and differentiation of the implanted cells are essential to guarantee
a potentially beneficial effect. Post-mortem immunohistochemical analyses 6 weeks
post-implantation indicated that in 70% of R6/2-undiff and 50% of R6/2-diff animals
some STROC05 cells survived six weeks post-implantation. A re-analysis of the behaviour
and MRI results indicated that exclusion of animals without surviving cells did not
significantly affect outcome (Additional file
4: Figure S4). Surviving cells were mostly confined to the injection tract (Figure
5A), with a select few showing a limited migration in the corpus callosum. In the left
hemisphere of R6/2-undiff mice, only 161.2 ± 46.8 STROC05 cells survived, whereas
81.9 ± 34.16 cells survived in R6/2-diff animals (Figure
5B). However, given the wide variability within each group, a statistically significant
difference between these two types of implants in terms of cell survival could not
be detected. Despite the generally poor cell survival, a small number of STROC05 cells
expressed Fox3 in both the R/6-undiff (1.2%) and R6/2-diff (2%) conditions (Figure
5C&D). DARPP-32 was not detected in any of the implanted cells. Almost all implanted
cells were GFAP-positive (Figure
5E) suggesting that predominantly astrocytic cells survived, whereas neurons did not.
Therefore, most implanted cells did not survive by 6 weeks post-implantation and pre-differentiation
of STROC05 cells did not increase the presence of neuronal cells post-grafting.

Additional file 4.Figure S4. Re-analysis of the main outcome measures. As some animals had no graft survival,
it is conceivable that this would affect the group outcome measure. Therefore we reanalysed
the data excluding these animals. The analysis containing all animals is presented
on the left and the reanalysed data on the right. Exclusion of animals without graft
survival, however, did not make a difference to these results.

Figure 5.Survival of the transplanted cells. A small population of implanted cells survived (A). Human cells (human nuclear antigen + cells in pink) were mostly found within the
injection tract in the striatum. A select few individual cells were observed migrating
along the corpus callosum. Stereological cell counts revealed no significant difference
between undifferentiated and differentiated cell implantation (B). However, cell survival was very variable with some animals having no surviving
cells. Neuronal differentiation, as determined by FOX3 staining, of implanted cells
was very poor (C&D). Most implanted cells differentiated into astrocytes (E), whereas others did neither express markers of neurons nor astrocytes. (Scale bar
200 μM).

Discussion

Cell therapy for Huntington’s disease is potentially an important intervention to
delay, stabilize and/or improve impairments. These therapeutic effects are well documented
in animal models, but more limited, albeit positive, evidence is available in patients
with Huntington’s disease that received fetal tissue transplants
[43]. However, in the present study, the STROC05 human neural stem cell line in the R6/2
mouse model of HD did not promote recovery. It is important to recognize that a multitude
of requirements need to be met for this therapy to be successful and several explanations
need to be considered to account for our results: 1) STROC05 cells are not efficacious
in HD, 2) insufficient cells survived to promote recovery, 3) there was an insufficient
neuronal/DARPP-32 differentiation of cells, and 4) it is also conceivable that the
R6/2 model might be too aggressive to evaluate hNSC as a restorative treatment.

Lack of efficacy and poor cell survival

Therapeutic efficacy in Huntington’s disease is considered to be associated with a
decrease in neurodegeneration, as well as a replacement of lost striatal DARPP-32+
GABAergic output neurons. An intra-striatal injection of fetal-derived neural progenitors/stem
cells
[2,13], NSC lines
[7,8], as well as mesenchymal cells
[44] produces an improvement in behavioural impairment. Even an intravenous injection
of mesenchymal cells can achieve improvements in Huntington’s disease with only a
small fraction of cells penetrating the brain
[45]. However, human neural stem cells from the STROC05 neural stem cell line did not
improve outcome in the R6/2 mouse model of HD.

It is therefore important to consider why STROC05 cells did not improve outcome. Foremost
of all, survival of cells after implantation was rather poor with only 161 human cells
surviving in one hemisphere. Although there have been reports of behavioural changes
with 124 cells surviving in stroke
[46], most efficacious studies using cell implantation in Huntington’s disease report
survival rates of 2 × 104 cells
[2]. Interestingly, STROC05 survival in the 3NPA rat model of HD resulted in 2.5 × 104 cells surviving at 3 months. It is therefore conceivable that either the progressive
pathology or the mouse host are factors that affect the long-term survival of these
cells. Improving cell survival in a mouse host will be key to establishing whether
the poor cell survival is the reason for the lack of efficacy. While there was no
evidence here of graft rejection, it is conceivable that an early immune response
could have affected cell survival and hence efficacy. If this were the case, administration
of immunosuppressants and anti-inflammatory treatment would be expected to improve
graft survival and potentially provide sufficient cell survival to promote recovery.

Although the survival of cells is thought to be essential to establish recovery by
means of intracerebral hNSC implantation, the lack of differentiation of STROC05 cells
might also preclude recovery. Especially, the differentiation of cells into striatal
DARPP-32+ GABAergic output neurons has been considered to be directly linked to the
degree of functional recovery
[47,48]. One approach to increase the number of DARPP-32+ neurons from implanted cells is
to direct their differentiation prior to injection. This can either be achieved using
chemical factors or genetic engineering
[9,18,20,49]. Although the hedgehog agonist purmorphamine here increased the differentiation of
STROC05 cells into DARPP-32+ neurons over 3 weeks in vitro without affecting viability when these cells are re-suspended, none of the cells
had survived for 6 weeks post-implantation. It is conceivable that this is a reflection
of the overall poor survival of cells, but it would be reasonable to expect that some
improvement in neu-ronal survival of implanted cells could be expected after implantation
of pre-differentiated cells. Nevertheless, this was not the case with an equally low
neuronal differentiation in the undifferentiated and differentiated cell groups. This
is in stark contrast to other reports where pre-differentiated cells exhibited good
survival with an improvement in the survival of DARPP-32 cells
[9,18,49]. There is indeed evidence that pre-differentiation of cells makes them especially
vulnerable to apoptosis
[50]. Improving overall cell survival might therefore also potentially increase the survival
of pre-differentiated cells, but as in Parkinson’s disease additional survival factors
(e.g. BDNF, GDNF) might be required to ensure the long-term survival and integration
of these neurons
[51,52].

Apart from poor cell survival and differentiation, it is plausible that, even if these
issues are overcome, this cell line is not efficacious in Huntington’s disease. If
this would indeed be the case, this cell line would provide an indispensable “therapeutic
control” condition against which mechanisms of efficacious cells could be compared.
Nevertheless, it is also conceivable that this cell line could provide efficacious
results if implanted under different experimental conditions.

Choosing an appropriate animal model of Huntington’s disease

STROC05 cells might be efficacious for Huntington’s disease, but it is possible that
testing them in the R6/2 model does not reflect their therapeutic potential. The R6/2
model rapidly manifests behavioural impairments, as well as regional brain atrophy.
This rapid progression of disease might be appropriate for screening pharmacological
agents that exert immediate effects, but the time window might be too short and aggressive
to evaluate the efficacy of neural progenitor/stem cells. Mouse models that develop
neuronal loss over a protracted time course, such as the YAC72
[53] or HDH(CAG)150[54] might hence provide more appropriate conditions to establish the therapeutic efficacy
of intracerebral cell implantation. Neural progenitor/stem cell implantation typically
takes several weeks before therapeutic effects are evident. Therefore when R6/2 mice
are almost moribund, implanted cells are expected to exert their effect and the disease
might have progressed too far at this stage for any efficacy to be apparent. Additionally,
the progression of the disease could impact on the cell’s survival
[55]. Similar observations were evident in a previous study in R6/2 mice using fetal primary
tissue grafts, where there was sufficient graft survival, but no meaningful therapeutic
efficacy
[56], although the same type of graft provided a significant improvement in neurotoxin-induced
lesions modelling Huntington’s disease
[13]. A similar difference in behavioural recovery between neurotoxic lesions in the mouse
and the R6/2 were also observed after an intrastriatal injection of mesenchymal stem
cells
[57]. Merely implanting neural progenitor/stem cells in transgenic mice might hence be
insufficient to achieve therapeutic efficacy.

A combination of treatments for various aspects of the disease might be needed for
implanted cells to be efficacious. For instance, an injection of only mouse neural
progenitors did not maintain motor function in N171-82Q transgenic mice, but if these
same progenitors were engineered to also secrete GDNF, they provided a therapeutic
benefit
[58]. In the R6/2 mice, therapeutic efficacy was also achieved with NSCs, but only if
these were administered in conjunction with a retardation of CAG aggregate formation
using trehalose
[59]. Transgenic mice therefore are likely to be appropriate models for establishing therapeutic
efficacy in Huntington’s disease, but a combinatorial approach that concurrently impacts
on different disease mechanisms might be needed to progress cell implantation as a
treatment strategy. Having to target multiple mechanisms of the disease, as well as
supplying novel cells to the brain, are likely to be a better reflection of the clinical
condition than expecting neural progenitor/stem cells to be sufficiently efficacious
to avert a further deterioration of patients.

Conclusions

Neither the implantation of undifferentiated, nor pre-differentiated human NSCs promoted
behavioural benefits or attenuated the on-going neurodegenerative process. This is
likely due to a combination of factors, most importantly cell survival was insufficient
to impact on the progression of the disease, but the life-span of R6/2 mice might
also be too short to appropriately evaluate neural progenitors/stem cells. More chronic
transgenic models are likely to be better in evaluating these therapies. However,
implantation of cells by themselves is unlikely to be sufficiently efficacious to
promote recovery, but rather a combination of multiple treatments will be required
to provide a truly efficacious therapy that can impact on the clinical condition.

Competing interests

MM previously received financial and personnel support from ReNeuron Ltd to study
the efficacy of a hNSC line in a rat model of stroke.

Acknowledgments

This study was funded through a translational stem cell grant by the UK Medical Research
Council (G0802552 & G0800846) and a PhD studentship by the Egyptian Government (MM45/07).
The authors would like to thank Dr Anthony Vernon for assistance with the 3-NPA study,
as well as Dr Po-Wah So, the manager of the preclinical imaging unit at KCL, and the
British Heart Foundation who funded the MRI scanner.